Nucleases, including zinc finger nucleases and homing endonucleases such as I-SceI, that are engineered to specifically bind to target sites have been shown to be useful in genome engineering in basic research and in the pharmaceutical and biotechnology applications. For example, zinc finger nucleases (ZFNs) are proteins comprising engineered site-specific zinc fingers (with engineered recognition regions) fused to a nuclease domain. Such ZFNs have been successfully used for genome modification in a variety of different species. See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and International Publication WO 07/014,275, the disclosures of which are incorporated by reference in their entireties for all purposes. These ZFNs can be used to create a double-strand break (DSB) in a target nucleotide sequence, which increases the frequency of donor nucleic acid introduction via homologous recombination at the targeted locus (targeted integration) more than 1000-fold. In addition, the inaccurate repair of a site-specific DSB by non-homologous end joining (NHEJ) can also result in gene disruption. Nucleases can be used for a wide variety of purposes such as for cell line engineering as well as for therapeutic applications.
Efficiency of nuclease activity can be influenced by a variety of factors such as accessibility of the target and the quality of the binding interaction between the nuclease and its target nucleic acid. To increase the success rate of nuclease driven genomic modifications, researchers often have to resort to introducing selectable markers during donor integration in order to be able to select variants that have had modifications from those that have not been modified (see, for example, U.S. Pat. No. 6,528,313). For a number of applications, use of selectable markers is not desirable as this technique leaves an additional gene or nucleic acid sequence inserted into the genome.
Thus, there remains a need for compositions and methods for increasing nuclease activity to allow for more efficient use of these powerful tools.